This proposal describes experiments to investigate the molecular mechanism of pathogenesis in prion diseases. It is built upon the hypotheses that the primary pathogenic event involves a specific interaction between the native and disease forms of the protein, and that this interaction destabilizes a particular sub-domain of the native structure. In order to test this hypothesis, in vitro spectroscopic studies will be performed on both the native protein, and mutants designed to inexorably identify the site of interaction. The specific aims of the proposal include studying the unfolding and conversion of the native recombinant protein, using site-directed mutagenesis to produce the key mutants, and performing the same folding/unfolding studies on each of these mutant proteins. Elucidation of molecular-level details will be afforded by fluorescence circular dichroism, infrared absorption and Ramon spectroscopies. The overall goal of this research is to provide the information necessary to begin rational design of molecular inhibitors of prion assembly. The overall goal of this research is to provide the information necessary to begin rational design of molecular inhibitors of prion assembly.